Viral infections remain a significant cause of treatment failure in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). Recipients of cord blood (CB) or HSC from virus naive donors are at particular risk since their grafts contain no virus-specific memory T-cells. Antiviral drugs are effective only for some viruses, and most have significant toxicities. Adoptive transfer of virus-specific cytotoxic T lymphocytes (CTL) from the stem cell donor has proved safe and highly effective, but is available only for recipients of grafts from virus-experienced donors, thereby excluding those patients at highest risk. This lack of an effective strategy to activate and expand virus-specific T-cells from CB and naive donor T-cells has been a major obstacle to extending the approach to these high-risk recipients, whose continued prolonged morbidity and high mortality from viral diseases substantially reduces the cost:benefit ratio of the transplant procedure. We have developed a novel approach that effectively expands CTL specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenoviruses (Ad) from naive CB T-cells. We expressed the CMVpp65 antigen from an Ad vector in dendritic cells and used them together with T-cell enhancing cytokines to activate T-cells specific for pp65 and Ad vector-derived virion proteins. Subsequent restimulation with CB-derived EBV-transformed lymphoblastoid cell lines transduced with the same vector also reactivated an EBV-specific T-cell component, so that the final T-cell product contained T-cells that were specific for all three viruses, and recognized multiple viral epitopes. We produced and validated the function of the virus-specific CTL from the 20% fraction of cryopreserved CB that will be available to us for our proposed phase I clinical trial of these cells. We now hypothesize that virus-specific CTL prepared from virus-naive CB (Aim 1) will restore antiviral immunity and reduce viral infection in CBT recipients (Aim 2). In these aims we will discover (i) if administration of CB-derived T-cells with multiple viral specificities can produce immune reconstitution to all three viruses, or if there is a consistent hierarchy of recovery, favoring one viral specificity over the others and (ii) whether viral infections will be prevented or controlled after adoptive transfer of CB-derived CTL. Aim 3 will determine whether the mechanisms by which cord blood T-cells respond to latent viral antigens are associated with their naivety or developmental stage. Our studies will show whether this approach can consistently produce effective CTL directed to three of the commonest pathogenic viruses after CBT and whether this approach has the potential to reduce morbidity and mortality after transplant. RELEVANCE (See instructions):